*NCBI Gene # or RefSeq#:TDR Targets ID: 25954 *Protein ID (NP or XP #) or Wolbachia#: ID: TriTrypDB / LmjF06.0860GeneDB / LmjF06.0860 *Organism (including strain):Leishmania major Etiologic Risk Group (see link below): Risk Group 2 (Parasitic Agents) *Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Leishmania Major can cause a disease called Zoonotic cutaneous leishmaniasis which can occur among people who live in rural areas and is distinguished by skin lesions that can become inflamed. Necrosis sores may develop followed by hypersensitivity and involvement of immune complexes. This is part of the healing process and contributes to strong specific immunity. Though Zoonotic cutaneous leishmaniasis has been known to heal spontaneously, a new drug called miltefosine has been tested as efficient and safe in several clinical trials.
Essentiality of this protein: 6 days (this was for parasitic
African trypanosome)
Moreover, thymidine addition to the culture media did not help in obtaining null parasites, suggesting that the dhfr-ts gene may be essential for T. cruzi survival in vitro. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3236718/).
--Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Enzyme concentration was determined spectrophotometrically by following absorbance at 280nm. The extinction coefficient for P. falciparum TS-DHFR is 83740 M−1cm−1, and for L. major TS-DHFR is 69955 M−1 cm−1. DHFR steady-state activity was assessed by reacting enzyme with H2folate and NADPH, and following absorbance at 340 nm, which decreases as NADPH is depleted to form NADP+. An extinction coefficient of −12.8 mM−1 cm−1 was used for the DHFR reaction.
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
Chain A, Trypanosoma Cruzi Dihydrofolate Reductase-Thymidylate Synthase Complexed With Nadph, Dump And C-448 Antifolate
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 98%
---- Max % Identities: 348/523(67%)
---- % Positives: 403/523(77%)
---- Chain used for homology: Chain A
Current Inhibitors:
10-PROPARGYL-5,8-DIDEAZAFOLIC ACID
METHOTREXATE
NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
2'-DEOXYURIDINE 5'-MONOPHOSPHATE Expression Information (has it been expressed in bacterial cells):
However, due to its weaker contribution (relative to DHFR-TS; Table III), PTR1 overexpression by gene amplification is apparently necessary to provide sufficient activity
Purification Method: Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSRAAARFKIPMPETKADFAFPSLRAFSIVVALDMQHGIGDGESIPWRVPEDMTFFKNQT
TLLRNKKPPTEKKRNAVVMGRKTWESVPVKFRPLKGRLNIVLSSKATVEELLAPLPEGQR
AAAAQDVVVVNGGLAEALRLLARPLYCSSIETAYCVGGAQVYADAMLSPCIEKLQEVYLT
RIYATAPACTRFFPFPPENAATAWDLASSQGRRKSEAEGLEFEICKYVPRNHEERQYLEL
IDRIMKTGIVKEDRTGVGTISLFGAQMRFSLRDNRLPLLTTKRVFWRGVCEELLWFLRGE
TSAQLLADKDIHIWDGNGSREFLDSRGLTENKEMDLGPVYGFQWRHFGADYKGFEANYDG
EGVDQIKLIVETIKTNPNDRRLLVTAWNPCALQKMALPPCHLLAQFYVNTDTSELSCMLY
QRSCDMGLGVPFNIASYALLTILIAKATGLRPGELVHTLGDAHVYRNHVDALKAQLERVP
HAFPTLIFKEERQYLEDYELTDMEVIDYVPHPAIKMEMAV
*length of your protein in Amino Acids: 520aa Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite 58688.6
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 69955 Abs 0.1% (=1 g/l) 1.192, assuming all pairs of Cys residues form cystines
*GC% Content for gene: 36% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to. Primer design results for 'tail' primers (this is just 2 sequences):
dihydrofolate reductase-thymidylate synthase
*NCBI Gene # or RefSeq#: TDR Targets ID: 25954
*Protein ID (NP or XP #) or Wolbachia#: ID: TriTrypDB / LmjF06.0860 GeneDB / LmjF06.0860
*Organism (including strain): Leishmania major
Etiologic Risk Group (see link below): Risk Group 2 (Parasitic Agents)
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Leishmania Major can cause a disease called Zoonotic cutaneous leishmaniasis which can occur among people who live in rural areas and is distinguished by skin lesions that can become inflamed. Necrosis sores may develop followed by hypersensitivity and involvement of immune complexes. This is part of the healing process and contributes to strong specific immunity. Though Zoonotic cutaneous leishmaniasis has been known to heal spontaneously, a new drug called miltefosine has been tested as efficient and safe in several clinical trials.
Essentiality of this protein: 6 days (this was for parasitic
African trypanosome)
Moreover, thymidine addition to the culture media did not help in obtaining null parasites, suggesting that the dhfr-ts gene may be essential for T. cruzi survival in vitro. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3236718/).
Complex of proteins? No
Druggable Target: 1
*EC#: 1.5.1.3
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.5.1.3
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Enzyme concentration was determined spectrophotometrically by following absorbance at 280nm. The extinction coefficient for P. falciparum TS-DHFR is 83740 M−1cm−1, and for L. major TS-DHFR is 69955 M−1 cm−1. DHFR steady-state activity was assessed by reacting enzyme with H2folate and NADPH, and following absorbance at 340 nm, which decreases as NADPH is depleted to form NADP+. An extinction coefficient of −12.8 mM−1 cm−1 was used for the DHFR reaction.
http://www.brenda-enzymes.org/literature/lit.php4?e=1.5.1.3&r=685197
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/dihydrofolatereductase.pdf
-- link (or citation) to paper that contains assay information
http://www.jbc.org/content/272/21/13883.long
-- List cost and quantity of substrate reagents and supplier
Tris-HCl-------------$49.00 (100g)-------T5941
NADPH-------------$99.90 (25mg)--------N5130
Sodium Acetate--$44.10 (500g)--------S8750
Sigma Aldrich Supplier
-- PDB # or closest PDB entry if using homology model:
Chain A, Trypanosoma Cruzi Dihydrofolate Reductase-Thymidylate Synthase Complexed With Nadph, Dump And C-448 Antifolate
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 98%
---- Max % Identities: 348/523(67%)
---- % Positives: 403/523(77%)
---- Chain used for homology: Chain A
Current Inhibitors:
10-PROPARGYL-5,8-DIDEAZAFOLIC ACID
METHOTREXATE
NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
2'-DEOXYURIDINE 5'-MONOPHOSPHATE
Expression Information (has it been expressed in bacterial cells):
However, due to its weaker contribution (relative to DHFR-TS; Table III), PTR1 overexpression by gene amplification is apparently necessary to provide sufficient activity
Purification Method:
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSRAAARFKIPMPETKADFAFPSLRAFSIVVALDMQHGIGDGESIPWRVPEDMTFFKNQT
TLLRNKKPPTEKKRNAVVMGRKTWESVPVKFRPLKGRLNIVLSSKATVEELLAPLPEGQR
AAAAQDVVVVNGGLAEALRLLARPLYCSSIETAYCVGGAQVYADAMLSPCIEKLQEVYLT
RIYATAPACTRFFPFPPENAATAWDLASSQGRRKSEAEGLEFEICKYVPRNHEERQYLEL
IDRIMKTGIVKEDRTGVGTISLFGAQMRFSLRDNRLPLLTTKRVFWRGVCEELLWFLRGE
TSAQLLADKDIHIWDGNGSREFLDSRGLTENKEMDLGPVYGFQWRHFGADYKGFEANYDG
EGVDQIKLIVETIKTNPNDRRLLVTAWNPCALQKMALPPCHLLAQFYVNTDTSELSCMLY
QRSCDMGLGVPFNIASYALLTILIAKATGLRPGELVHTLGDAHVYRNHVDALKAQLERVP
HAFPTLIFKEERQYLEDYELTDMEVIDYVPHPAIKMEMAV
*length of your protein in Amino Acids: 520aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite 58688.6
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 69955 Abs 0.1% (=1 g/l) 1.192, assuming all pairs of Cys residues form cystines
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGGCTTCTACTGGTGAAACTATATGTAACAATGGCCAAAGCGCAGTTTCAGTAACAAGGAGGAGGAGTT ACCAAGTTGTAATAGCGGCGACAAGAGACATGGGTCTTGGTATGGACATGAAACTTCCTTGGGATCTACC TTCAGAGTATCAGTTTTTCCAAGATGTTACAACTAGGACATCCGATCCCACGAAGAGGAACGCGACTATA ATGGGAAGAAAATCATGGGAATCTACTCCTCTAGAGATTCGTCCTCTTCCTGGTCGGCTCAATATTGTCT TGACAAAGTCTAGCTGCCACAACATTGCCATTGATGAGAATGTACTGGTGTCTAGTAGCATGGAATCGGC TCTTGAACTTCTAGCCACTGAGCCCTATTCCTTGTCCATTGAGAAGGTCTTTGTCATAGGAGGCGGCGAG TTGTTAAGGTTTGCATCTTCTTTCCACAAGGGTTTTGTTCTTTTTCTAAAAACACATGTTTATGTCTTGA TATAAGCGTGTCATGCGATGTTTGAGACTTTAGACCTTGAGGTTTGATGTCTTGATTTGATTGATGGTAC AATGATTGAAGTTCTTTTAAGGCCTTAAGTTAGATGGTTGAAGTTTAGTACTGTGAATGAGAGAATGATT AATTGTTGTGGTTTCTCTACAGGAATTATATGAATGCATCCATCTGTGATGCTATCCACCTAACCGAAAT CGATATAAGCGTGCCATGTGACGCATTTGCACCGAGAGTGGATACTTCTCTGTACCGTCCGTGGTACTCA TCATTTCCAGTTGTGGAAAATGGAATTCGGTATAGTTTCAACACTTATGTCCGAAGAAAAGATGCCATTG TTGGCTCCGGTGAAAAGAAAAGTGTTGCTGAGTCAGATTTGAAGGAGTACTCGTTTTTGCCTAAGATGGT TTTCGAGAGGCATGAGGAGTTTGGTTACTTGAATCTTGTTCAAAACATTATATCTAGTGGAGACATGAAT GACAATAGTACTCTTTCTAAATTTGGCTGTCAGGTGAGAGCTACACTCAATCTTTGTTAGTATCTTTCTC CTTTAATTGAGCTTACATGTTCTTTGTTCTTGATTTGGTTCATTTACAGATGCGGTTCAATCTGCGCAAG ACTTTCCCGCTTCTCACGACCAAGGCATGTCTTCTTTTATTCACATCTCTTATCAAAGTAGTTCACCTTT ATTTGATCACAGAGATAAATTTTGTCGGTTTTTGTACCCGTTGCCTTTCAGAAAATATTCTGGCTTGGTG TTGTAGAGGAAATACTACAACTTATCAGTGGTTCAAACAATCCCAAGGTAAAACCAAGAGAGACCTGTGC TACTACACAGTAAATGACAATGATCATGCCCAAAATTCTTGGGTTAAAGATATGTTTTCTTTGTTTGATA TATTGATAAAAAACCGAGAATGTAACATACTAGATTTTAACCCGCGGTACACCGCGGAGACAATTAATTT TTTAATATATATAAAAGTTTGCAAATTGTATCTATATATAAAATATTTTTATTTTAAAGTTTACAATTGT TATTAAATAATGTCCTGTCAAACTCGTCTCGTAAAAACCTATTTATTTTATTAATGTTGATTTTATTTAT GATATGTTTATAAATCATTGTATAAATATTTTGTAATTTTGTAGTAATTAACTGCTATCAAATATTTCTG CAGTTTGATGCTTATAATCGAATTGTTATAGTCATGAAAAAAGCAATATCAAAAATGATAATTTTGTAGT GTTTAAATGATATTAAATATTTTGGAAGTTTTATTTTAGTAACTAATTGTGTAGTTAATGTGGAGAGATT TTGGGAAGATAGTTAAATGTCAAATATTTAATTCGAAGATTTACTTTCTAATTATCAAAAATAATTATTA AATGTCAGTGGCAGCTACTGTAAATAAGTCCCAACTTGATGATTTATTTCACAAAATGGCTGCAAAAATG TATATATAGATTATATCTCATATGTTCTTTCAGGAAAACGGTAGTCATATATGGGACACCGATGAGGCAA AAGAATATCTTGACAGGCAAGCACAAACTTTAGAATTTAAAATGCACTTTTACTTATACAAAACTCACAT CCATGCCTATGTTCCAGTTTCGGAGTGAATGCCACCGAAGAAGATGGAGACAACCCATTCTTGCATGGAC TTCACTGGAAACATTGTGATGCTAGGTTTGTGATGTCTCTTCTTTGGATGTTTGTCTCATATGCTGTGCC TCTTTTGACTGATTGGTTTTACATTTTACCAGTCAAGAATTCAGTCAGCTCTCTGATGTTATAAACAAAA TAAAGAACAATCCTCATGATCAAAGGATCATGCTTGCCGCTTGTAATCCATTAGATTTCAAGTTGTCGGT ATCTCCTTGTCATACGTTCACACAGGTTAGCACGTTTTTCATCTTGAAACTTGTTACATTTCCATTGTAA AATCATTTCAACGTTTCAAGATATACAAAACTGAAAATGATTTGGTTTTTGGTTGTTGTTGCCAGTTCTA TGTGGCAAATGGTGAAGTTTCTTGTCAAATATACCAAAGCTCAACCGAAGCGAGTATCGGGATTCCTTTC AGTATCGCTACATACTCTCTTTTGACATGCATCATCGCTCATGTTTGCGGTATGTACACATTTTATTAGC TCTTGAGTCTAAAGTAAAAGAGTTGAAACCTTTGTATGGTTTCTTGTCTTGACAGATCTTGGAGCTGGTG ATTTCATCCATGTGATTGGACAAGCCTATATTAACAAAGCTCACGTCAAGGCTATACAAAAACAGCTTCA AATCTCCCCTAAACCCTTCCCGGTAAGTACTAAATATATTTTGCTCTCTCTCAAATCAGGAAACAATATA ACTTGTGTGTCTCTAATGTATTAACGGTAAAAACAGATTTTGAAAATCAACCCTGAGAAAAAGAAGATGG ACAACTTTGAGGCCTCTGATTTGGAACTCATGAGGATATAA
*GC% Content for gene: 36%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Sources:
NIAID Emerging and Re-emerging Infectious Diseases – Group I, II, and III
http://www.niaid.nih.gov/topics/emerging/pages/list.aspx
NIAID Biodefense – Group III: Category A, B, and C Priority Pathogens
http://www.niaid.nih.gov/topics/BiodefenseRelated/Biodefense/research/Pages/CatA.aspx
TDR targets webpage www.tdrtargets.org
J Craig Venter Institute – New Infectious Diseases Targets http://gsc.jcvi.org/projects.html
Neglected and Tropical Diseases (PLoS One) - http://www.plosntds.org/static/scope.action
http://www.niaid.nih.gov/LabsAndResources/resources/dmid/brc/Pages/awards.aspx
http://pathogenportal.net/portal/portal/PathPort/Home
EUPATH: Eukaryotic Pathogens Database Resources: http://eupathdb.org/eupathdb/
Etiologic Risk Group Categories (for pathogens):
http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
BRENDA webpage for enzymes – can search by substrate by doing advanced search
http://www.brenda-enzymes.org/
SIGMA - chemical supplier- http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/assay-library/ec-number.html
http://blast.ncbi.nlm.nih.gov/Blast.cgi - Protein Similarity
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